Chemical cross-linking of a variety of green fluorescent proteins as Förster resonance energy transfer donors for Yukon orange fluorescent protein: A project-based undergraduate laboratory experience

2018 ◽  
Vol 46 (5) ◽  
pp. 516-522 ◽  
Author(s):  
Mira K. Marchioretto ◽  
Jacqueline T. Horton ◽  
Calvin A. Berstler ◽  
Joseph B. Humphries ◽  
Isaac J. Koloditch ◽  
...  
Keyword(s):  
Fluorescent Protein ◽  
Fluorescent Proteins ◽  
Protein A ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Green Fluorescent Proteins ◽  
Orange Fluorescent Protein ◽  
Undergraduate Laboratory ◽  
Green Fluorescent ◽  
Chemical Cross Linking

scholarly journals An endogenous green fluorescent protein–photoprotein pair in Clytia hemisphaerica eggs shows co-targeting to mitochondria and efficient bioluminescence energy transfer

Open Biology ◽  
2014 ◽  
Vol 4 (4) ◽  
pp. 130206 ◽  
Author(s):  
Cécile Fourrage ◽  
Karl Swann ◽  
Jose Raul Gonzalez Garcia ◽  
Anthony K. Campbell ◽  
Evelyn Houliston
Keyword(s):  
Energy Transfer ◽  
Fluorescent Protein ◽  
Fluorescent Proteins ◽  
Resonance Energy Transfer ◽  
Green Light ◽  
Resonance Energy ◽  
Green Fluorescent Proteins ◽  
Parallel Acquisition ◽  
Green Fluorescent

Green fluorescent proteins (GFPs) and calcium-activated photoproteins of the aequorin/clytin family, now widely used as research tools, were originally isolated from the hydrozoan jellyfish Aequora victoria . It is known that bioluminescence resonance energy transfer (BRET) is possible between these proteins to generate flashes of green light, but the native function and significance of this phenomenon is unclear. Using the hydrozoan Clytia hemisphaerica , we characterized differential expression of three clytin and four GFP genes in distinct tissues at larva, medusa and polyp stages, corresponding to the major in vivo sites of bioluminescence (medusa tentacles and eggs) and fluorescence (these sites plus medusa manubrium, gonad and larval ectoderms). Potential physiological functions at these sites include UV protection of stem cells for fluorescence alone, and prey attraction and camouflaging counter-illumination for bioluminescence. Remarkably, the clytin2 and GFP2 proteins, co-expressed in eggs, show particularly efficient BRET and co-localize to mitochondria, owing to parallel acquisition by the two genes of mitochondrial targeting sequences during hydrozoan evolution. Overall, our results indicate that endogenous GFPs and photoproteins can play diverse roles even within one species and provide a striking and novel example of protein coevolution, which could have facilitated efficient or brighter BRET flashes through mitochondrial compartmentalization.

Fluorescent Indicators for Calcium Based on Green Fluorescent Proteins (GFPs) and Calmodulin

1998 ◽  
Vol 4 (S2) ◽  
pp. 446-447
Author(s):  
A. Miyawaki ◽  
J. Llopis ◽  
R. Heim ◽  
J.M. McCafFery ◽  
J.A. Adams ◽  
...  
Keyword(s):  
Fusion Proteins ◽  
Fluorescent Protein ◽  
Fluorescent Proteins ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Green Fluorescent Proteins ◽  
Fluorescent Indicators ◽  
Calmodulin Binding ◽  
Green Fluorescent ◽  
Partner Proteins

Cytosolic and organellar free Ca2+ concentrations are very dynamic; they are often extremely localized and hard to measure. To overcome this problem we have constructed new fluorescent indicators for Ca2+ that are genetically encoded without cofactors and are targetable to specific intracellular locations.Green fluorescent protein (GFP) is a spontaneously fluorescent protein from the jelly fish Aequorea victoria. Its cDNA can be concatenated with those encoding many other proteins, and the resulting fusion proteins are usually fluorescent and often preserve the biochemical functions and cellular localizations of the partner proteins. Mutagenesis has produced GFP mutants with shifted wavelengths of excitation or emission that can serve as donors and acceptors for fluorescence resonance energy transfer (FRET).Our new indicators consist of tandem fusions of a blue- or cyan-emitting mutant of GFP, calmodulin (CaM), the calmodulin-binding peptide Ml3, and an enhanced green- or yellow-emitting GFP. Binding of Ca2+ makes calmodulin wrap around the Ml3 domain, increasing the FRET between the flanking GFPs (Fig. 1).

scholarly journals Dynamic tuning of FRET in a green fluorescent protein biosensor

2019 ◽  
Vol 5 (8) ◽  
pp. eaaw4988 ◽  
Author(s):  
Pablo Trigo-Mourino ◽  
Thomas Thestrup ◽  
Oliver Griesbeck ◽  
Christian Griesinger ◽  
Stefan Becker
Keyword(s):  
Green Fluorescent Protein ◽  
Protein Interactions ◽  
Fluorescent Protein ◽  
Fluorescent Proteins ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Structural Data ◽  
X Ray ◽  
Dynamic Tuning ◽  
Green Fluorescent

Förster resonance energy transfer (FRET) between mutants of green fluorescent protein is widely used to monitor protein-protein interactions and as a readout mode in fluorescent biosensors. Despite the fundamental importance of distance and molecular angles of fluorophores to each other, structural details on fluorescent protein FRET have been missing. Here, we report the high-resolution x-ray structure of the fluorescent proteins mCerulean3 and cpVenus within the biosensor Twitch-2B, as they undergo FRET and characterize the dynamics of this biosensor with B02-dependent paramagnetic nuclear magnetic resonance at 900 MHz and 1.1 GHz. These structural data provide the unprecedented opportunity to calculate FRET from the x-ray structure and to compare it to experimental data in solution. We find that interdomain dynamics limits the FRET effect and show that a rigidification of the sensor further enhances FRET.

scholarly journals Intramolecular Fluorescence Resonance Energy Transfer between Fused Autofluorescent Proteins Reveals Rearrangements of the N- and C-terminal Segments of the Plasma Membrane Ca2+ Pump Involved in the Activation

2007 ◽  
Vol 282 (49) ◽  
pp. 35440-35448 ◽  
Author(s):  
Gerardo R. Corradi ◽  
Hugo P. Adamo
Keyword(s):  
Plasma Membrane ◽  
Energy Transfer ◽  
Fluorescence Resonance Energy Transfer ◽  
Fluorescent Proteins ◽  
Average Distance ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Green Fluorescent Proteins ◽  
Fluorescence Resonance ◽  
Green Fluorescent

The blue and green fluorescent proteins (BFP and GFP) have been fused at the N- and C-terminal ends, respectively, of the plasma membrane Ca2+ pump (PMCA) isoform 4xb (hPMCA4xb). The fusion protein was successfully expressed in yeast and purified by calmodulin affinity chromatography. Despite the presence of the fused autofluorescent proteins BFP-PMCA-GFP performed similarly to the wild-type enzyme with respect to Ca2+-ATPase activity and sensitivity to calmodulin activation. In the autoinhibited state BFP-PMCA-GFP exhibited a significant intramolecular fluorescence resonance energy transfer (FRET) consistent with the location of the fluorophores at an average distance of 45Å. The FRET intensity in BFP-PMCA-GFP decreased when the enzyme was activated either by Ca2+-calmodulin, partial proteolysis, or acidic lipids. Moreover, FRET decreased and became insensitive to calmodulin when hPMCA4xb was activated by mutation D170N in BFP-PMCA(D170N)-GFP. The results suggest that the ends of the PMCA are in close proximity in the autoinhibited conformation, and they separate or reorient when the PMCA achieves its final activated conformation.

scholarly journals The effect of proximity on the function and energy transfer capability of fluorescent protein pairs

2019 ◽  
Author(s):  
Jacob R. Pope ◽  
Rachel L. Johnson ◽  
W. David Jamieson ◽  
Harley L Worthy ◽  
Senthilkumar D. Kailasam ◽  
...  
Keyword(s):  
Energy Transfer ◽  
Protein Interactions ◽  
Fluorescent Protein ◽  
Fluorescent Proteins ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Protein Protein Interactions ◽  
Fluorescent Properties ◽  
Transition Dipole ◽  
Green Fluorescent

AbstractFluorescent proteins (FPs) are commonly used in pairs to monitor dynamic biomolecular events through changes in their proximity via distance dependent processes such as Förster resonance energy transfer (FRET). Many FPs have a tendency to oligomerise, which is likely to be promoted through attachment to associating proteins through increases in local FP concentration. We show here that on association of FP pairs, the inherent function of the FPs can alter. Artificial dimers were constructed using a bioorthogonal Click chemistry approach that combined a commonly used green fluorescent protein (superfolder GFP) with itself, a yellow FP (Venus) or a red FP (mCherry). In each case dimerisation changes the inherent fluorescent properties, including FRET capability. The GFP homodimer demonstrated synergistic behaviour with the dimer being brighter than the sum of the two monomers. The structure of the GFP homodimer revealed that a water-rich interface is formed between the two monomers, with the chromophores being in close proximity with favourable transition dipole alignments. Dimerisation of GFP with Venus results in a complex displaying ∼86% FRET efficiency, which is significantly below the near 100% efficiency predicted. When GFP is complexed with mCherry, FRET and mCherry fluorescence itself is essentially lost. Thus, the simple assumptions used when monitoring interactions between proteins via FP FRET may not always hold true, especially under conditions whereby the protein-protein interactions promote FP interaction.Abstract Figure

scholarly journals Far-red fluorescent tag for protein labelling

2002 ◽  
Vol 368 (1) ◽  
pp. 17-21 ◽  
Author(s):  
Arkady F. FRADKOV ◽  
Vladislav V. VERKHUSHA ◽  
Dmitry B. STAROVEROV ◽  
Maria E. BULINA ◽  
Yurii G. YANUSHEVICH ◽  
...  
Keyword(s):  
Fluorescent Protein ◽  
Fluorescent Proteins ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Sea Anemones ◽  
Practical Applications ◽  
In Vivo Labelling ◽  
Covalently Linked ◽  
Green Fluorescent

Practical applications of green fluorescent protein ('GFP')-like fluorescent proteins (FPs) from species of the class Anthozoa (sea anemones, corals and sea pens) are strongly restricted owing to their oligomeric nature. Here we suggest a strategy to overcome this problem by the use of two covalently linked identical red FPs as non-oligomerizing fusion tags. We have applied this approach to the dimeric far-red fluorescent protein HcRed1 and have demonstrated superiority of the tandem tag in the in vivo labelling of fine cytoskeletal structures and tiny nucleoli. In addition, a possibility of effective fluorescence resonance energy transfer ('FRET') between enhanced yellow FP mutant ('EYFP') and tandem HcRed1 was demonstrated in a protease assay.

scholarly journals Single-Molecule Studies on a FRET Biosensor: Lessons from a Comparison of Fluorescent Protein Equipped versus Dye-Labeled Species

Molecules ◽  
2018 ◽  
Vol 23 (12) ◽  
pp. 3105 ◽  
Author(s):  
Henning Höfig ◽  
Michele Cerminara ◽  
Ilona Ritter ◽  
Antonie Schöne ◽  
Martina Pohl ◽  
...  
Keyword(s):  
Conformational Change ◽  
Single Molecule ◽  
Fluorescent Dyes ◽  
Fluorescent Protein ◽  
Fluorescent Proteins ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Strong Increase ◽  
Single Molecule Level ◽  
Labeling Scheme

Bacterial periplasmic binding proteins (PBPs) undergo a pronounced ligand-induced conformational change which can be employed to monitor ligand concentrations. The most common strategy to take advantage of this conformational change for a biosensor design is to use a Förster resonance energy transfer (FRET) signal. This can be achieved by attaching either two fluorescent proteins (FPs) or two organic fluorescent dyes of different colors to the PBPs in order to obtain an optical readout signal which is closely related to the ligand concentration. In this study we compare a FP-equipped and a dye-labeled version of the glucose/galactose binding protein MglB at the single-molecule level. The comparison demonstrates that changes in the FRET signal upon glucose binding are more pronounced for the FP-equipped sensor construct as compared to the dye-labeled analog. Moreover, the FP-equipped sensor showed a strong increase of the FRET signal under crowding conditions whereas the dye-labeled sensor was not influenced by crowding. The choice of a labeling scheme should therefore be made depending on the application of a FRET-based sensor.

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